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Macromolecular recognition through electrostatic repulsion

H Bedouelle1, R Nageotte

  • 1Groupe d'Ingénierie des Protéines (CNRS URA 1129), Institut Pasteur, Paris, France.

The EMBO Journal
|June 15, 1995
PubMed
Summary
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Tyrosyl-tRNA synthetase uses Glu152 to reject incorrect tRNAs, preventing harmful amino acid misincorporations. Mutations altering this residue

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Aminoacyl-tRNA synthetases are crucial enzymes in genetic translation, ensuring accurate protein synthesis by attaching specific amino acids to their cognate transfer RNAs (tRNAs).
  • Loss of specificity in this process can lead to misincorporation of amino acids into proteins, potentially causing cellular dysfunction and disease.

Purpose of the Study:

  • To investigate the role of Glutamate 152 (Glu152) in tyrosyl-tRNA synthetase from Bacillus stearothermophilus in discriminating between cognate and non-cognate tRNAs.
  • To understand how alterations at residue 152 affect enzyme specificity, misaminoacylation, and cellular toxicity.

Main Methods:

  • Site-directed mutagenesis was employed to create 11 variants of Glu152 in tyrosyl-tRNA synthetase.
  • In vitro aminoacylation assays were performed to measure the misaminoacylation of non-cognate tRNAs (tRNA(Phe) and tRNA(Val)) with tyrosine.

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  • In vivo toxicity assays were conducted to assess the cellular impact of altered enzyme specificity.

    • Main Results:

    • Mutations at Glu152 resulted in a correlated increase in misaminoacylation of tRNA(Phe) and tRNA(Val) with tyrosine and increased cellular toxicity.
    • The degree of misaminoacylation and toxicity correlated with the change in the side chain's charge at position 152, from negative to uncharged to positive.
    • Aminoacylation of the cognate tRNA(Tyr) remained unaffected by the mutations at position 152.

    Conclusions:

    • Glutamate 152 plays a critical, purely negative role in tRNA discrimination by tyrosyl-tRNA synthetase.
    • This residue functions via electrostatic repulsion to prevent the binding and aminoacylation of non-cognate tRNAs.
    • The mechanism of tRNA discrimination involving electrostatic repulsion by Glu152 is evolutionarily conserved.